流感病毒糖蛋白糖链的作用和功能研究

流感病毒是近几年的研究热点之一.糖链在流感病毒生活周期中发挥重要作用,例如宿主细胞表面的唾液酸化糖链是病毒侵染细胞时的特异性受体,宿主决定的病毒糖蛋白糖链结构影响病毒的宿主范围和毒力.本文从糖组学角度综述糖链在甲型流感病毒生活周期中的重要作用,着重阐述病毒血凝素糖基化的影响因素及其对病毒宿主范围、毒力的影响和在病毒演化...     

基于MALDI-TOF质谱技术分析人宫颈癌 HeLa细胞N-糖链结构轮廓

以微量HeLa细胞(10⁷个)为对象, 经细胞裂解、还原羧甲基化、胰酶降解和Oasis-HLB柱提取分离得到总糖肽后, 用PNGase F酶解释放N-糖链. 对所得N-糖链用Sep-Pak C₁₈柱纯化后进行完全甲基化衍生, 再采用基质辅助激光解吸电离-飞行时间质谱(MALDI-TOF MS)分析HeLa细胞表面N-糖链的结构轮廓. 结果表明, 在获得的34种N-糖链中, 除高甘露糖型、二天线、三天线、四天线和五天线等N-糖链外, 还出现了在某种程度上与肿瘤发生转移相关的特殊平分型和Lewis结构. 利用MALDI-TOF MS技术可快速分析微量癌细胞表面N-糖链的结构轮廓, 为进一步寻找肿瘤糖链标志物及肿瘤的早期预防诊断提供技术支持.     

构筑“甜蜜的”纳米结构促进细胞内吞

复旦大学陈国颂教授等通过巧妙的高分子链结构设计,共价键与非共价相互作用的分级协同,精确地控制了糖功能基元在纳米粒子表面的分布,研究了糖功能基元分布对细胞内吞的影响.     

基于电喷雾电离质谱的人肝癌细胞HepG2与正常肝细胞L02的N-糖链的定性定量比较

以培养的原发性肝癌细胞HepG2和正常肝细胞L02为研究对象,采用细胞裂解液提取总蛋白,用PNGase F酶解释放N-糖链,以微晶纤维素柱结合石墨碳柱纯化分离N-糖链,通过电喷雾电离质谱(ESI-MS)和串联质谱(MS/MS)对N-糖链进行序列鉴定,以β-环糊精为内标对2种细胞系的N-糖链进行了定量比较分析.结果表明,在肝癌细胞系HepG2和正常细胞系L02中共检测到26种N-糖链,与L02相比,HepG2的大多数高甘露糖型糖链、唾液酸化糖链和岩藻糖基化糖链的数量都明显升高,其中有15种糖链在数量上具有极显著性差异(p0.01),1种糖链具有显著性差异(p0.05).本研究为进一步探索肝癌中各类N-糖链的表达特点及发现早期肝癌糖链标志物提供了参考.     

人肝癌细胞HepG2与正常肝细胞L02的Ο-糖链的比较分析

以培养的原发性肝细胞癌HepG2细胞和正常肝细胞L02为研究对象,用细胞裂解液提取总蛋白,然后采用Carlson还原性β-消除法释放O-糖链,以阳离子交换柱结合C18柱纯化分离O-糖链,用电喷雾电离质谱( ESI-MS)和串联质谱( MS/MS)对O-糖链进行序列鉴定,以β-环糊精为内标对2种细胞系的O-糖链进行定量比较分析.结果表明,在肝癌细胞系HepG2中检测到10种O-糖链,正常细胞系L02中检测到9种O-糖链,其中9种O-糖链是2种细胞系中共有的,但HepG2中存在癌细胞中特有的缩短的O-糖链N1A1( NeuAc-GalNAc, sialyl Tn 抗原). t检验结果表明, HepG2与L02相比,在检测到的10种O-糖链中有5种的含量具有极显著性差异(P<0.01),2种的含量具有显著性差异(P<0.05).     

人肝癌细胞HepG2与正常肝细胞L02的O-糖链的比较分析

以培养的原发性肝细胞癌HepG2细胞和正常肝细胞L02为研究对象, 用细胞裂解液提取总蛋白, 然后采用Carlson还原性β-消除法释放O-糖链, 以阳离子交换柱结合C₁₈柱纯化分离O-糖链, 用电喷雾电离质谱(ESI-MS)和串联质谱(MS/MS)对O-糖链进行序列鉴定, 以β-环糊精为内标对2种细胞系的O-糖链进行定量比较分析. 结果表明, 在肝癌细胞系HepG2中检测到10种O-糖链, 正常细胞系L02中检测到9种O-糖链, 其中9种O-糖链是2种细胞系中共有的, 但HepG2中存在癌细胞中特有的缩短的O-糖链N1A1(NeuAc-GalNAc, sialyl Tn 抗原). t检验结果表明, HepG2与L02相比, 在检测到的10种O-糖链中有5种的含量具有极显著性差异(P<0.01), 2种的含量具有显著性差异(P<0.05).     

基于质谱技术研究黑色素瘤血清特异性O-糖链

对接种和未接种B16黑色素瘤细胞的C57小鼠进行血清O-糖链比较糖组学研究,寻找黑色素瘤血清特异性O-糖链.小鼠血清10μL,β-消除反应释放O-糖链.反应混合物经石墨化炭黑固相萃取小柱(GCC SPE)分离纯化后,用于MALDI-Qit-TOF-MS分析.通过Launchpad软件采集并输出质谱数据,MATLAB进行数据解析,找到了10个稳定出现的差异糖链质谱峰.利用串联质谱分析了其中5个主要差异糖链的结构.     

基于寡糖代谢工程的正常和肿瘤细胞中Mucin型O-糖链的质谱定性定量比较分析

基于寡糖代谢工程结合质谱技术(MS结合MS/MS),对4种肿瘤细胞系和1种正常细胞系中的O-糖链进行了定性和相对定量比较分析.结果表明,4种肿瘤细胞系HeLa,SMMC-7721,HepG2和MCF-7中分别检测到19,11,6和5条O-糖链;在正常肝细胞系L02中检测到10条O-糖链.在对肿瘤和正常细胞系中表达的O-糖链进行定性和相对定量比较中发现,结构组成为N1,H1N1A1和H1N1A2的3种糖链在5种细胞系中均有表达;肿瘤细胞系表达的O-糖链的种类比正常细胞多,且岩藻糖基化和唾液酸化程度均高于正常细胞组.肿瘤细胞系中特有的O-糖链主要有岩藻糖化和唾液酸化修饰的Mucin型Core2结构糖链.MS/MS分析表明,其中岩藻糖基化修饰的O-糖链结构组成为H3N3F1A2,H4N4F1A2和H5N5F1A1,唾液酸化修饰的O-糖链结构组成为H5N4A1,H4N4A2和H5N5A2.     

微量样本中N-糖组分析技术的发展与应用

针对糖组学分析面临的初始样本量需求较高的技术挑战,该研究发展了一种微量样本N-糖链制备技术(GPAT),通过在移液枪头(Tip)中分别装填C_(18)和HILIC填料,实现了一站式原位蛋白消化、N-糖链释放和富集。与目前普遍采用的基于过滤辅助样品制备(FASP)技术的N-糖组分析策略相比,使用GPAT技术可以实现微量免疫球蛋白G(IgG)和人肝癌HepG2细胞提取蛋白的原位N-糖链的释放和富集,样本起始量减少90%,可以从10μg IgG和10μg的HepG2细胞蛋白中分别检测到20条和39条N-糖链。从6例健康人(3例男性,3例女性)尿液中提取的10μg尿蛋白中检测到49条N-糖链。该方法实现了微量复杂蛋白样本N-糖链简便、快速的定量分析策略,为进一步的糖组学方法推广应用奠定了基础。     

新型核壳型亲水聚合物-硅胶杂化填料的合成及其在蛋白质N-糖链富集中的应用

蛋白质的N-糖基化修饰与多种重要的生理、病理进程密切相关,是多种重大疾病诊断标志物研究的热点。由于糖蛋白本身多是低丰度表达的蛋白质,且糖链结构具有高度微不均一性,这使得蛋白质糖基化修饰的分析具有一定的挑战。本研究利用表面引发-原子转移自由基聚合(SI-ATRP)法,以带有双键的氨基葡萄糖为单体(GMAG),成功制备了新型核壳型亲水聚合物-硅胶杂化填料(pGMAG-SiO₂)。由于在硅胶表面引入致密的亲水聚合物层,该填料不仅保持了硅胶良好的机械强度,而且显著提高了其亲水性,因此非常适合作为亲水填料用于蛋白质的N-糖链富集。以麦芽七糖和鸡卵清蛋白的N-糖链为研究对象,考察了该填料对N-糖链的富集效果,并将该杂化填料成功用于人血浆中糖蛋白N-糖链的富集检测,共鉴定了47种糖型。以上结果表明,pGMAG-SiO₂填料对N-糖链具有较高的亲和性,可以用于N-糖链的高覆盖率鉴定。     

A note on the inactivation of influenza A viruses by solar radiation, relative humidity and temperature

We critically investigate the claim put forward by Sagripanti and Lytle ([2007] Photochem. Photobiol. 83, 1278-1282) that inactivation of influenza A virus by solar radiation can explain the seasonality of influenza epidemics. We correct an error in the Sagripanti and Lytle paper and show that changes in relative humidity and temperature affect influenza virus inactivation as strongly as variation in solar radiation. Furthermore, it appears unlikely that transmission in outdoor settings plays an important role during influenza outbreaks, because influenza A virus is sensitive to a wide range of environmental factors.     

Pharmacophore Mode lingand Virtual Screening to Design the Potential Influenza Virus Endonuclease Inhibitors

Influenza virus endonuclease is an attractive target for antiviral therapy in the treatment of influenza infection. The purpos e of this study is to design a novel antiviral agent with improved biological activities against the influenza virus endonuclease. In this study, chemical feature‐based 3D pharmacophore models were developed from 41 known influenza virus endonuclease inhibitors. The best quantitative pharmacohore model (Hypo 1), which consists of two hydrogen‐bond acceptors and two hydrophobic features, yields the highest correlation coefficient (R = 0.886). Hypo 1 was further validated by the cross validation method and the test set compounds. The application of this model for predicting the activities of 11 known influenza virus endonuclease inhibitors in the test set shows great success. The correlation coefficient of 0.942 and a cross validation of 95;% confidence level prove that this model is reliable in identifying structurally diverse compounds for influenza virus endonuclease inhibition. The most active compound (compound 1) from the training set was docked into the active site of the influenza virus endonuclease as an additional verification that the pharmacophore model is accurate. The docked conformation showed important hydrogen bond interactions between the compound and two amino acids, Lys 134 and Lys 137. After validation, this model was used to screen the NCI chemical database to identify new influenza virus endonuclease inhibitors. Our study shows that the to pranking compound out of the 10 newly identified compounds using fit value ranking has an estimated activity of 0.049 μM. These newly identified lead compounds can be further experimentally validated using in vitro techniques.     

FluClass: A novel algorithm and approach to score and visualize the phylogeny of the influenza virus using mass spectrometry

A novel computer algorithm FluClass has been developed to facilitate the phylogenetic classification of influenza virus using mass spectral data. FluClass accepts a DNA or protein-based phylogenetic tree as input and generates theoretical peptide mass lists for each node. An experimental mass spectrum from an influenza virus protein digest is then placed onto the phylogenetic tree using a novel random resampling function (Z-score) that allows the scoring of spectrum against both internal and leaf nodes. Testing of the algorithm using hemagglutinin protein sequences from human-host influenza viruses showed that the Z-score performs comparably to the Profound scoring method for the scoring of leaf nodes and is substantially better at scoring internal nodes. Scoring of internal nodes allows colorizations of nodes of the phylogenetic tree enabling the classification of the query spectrum to be rapidly visualized. Finally we demonstrate the utility of FluClass on experimental spectra from six strains. Given that mass spectrometry data can be generated rapidly for influenza virus proteins, FluClass provides a fast and direct method for phylogenetic analysis of influenza proteins.     

利用网络药理学方法研究热毒宁注射液抗流感病毒的分子作用机制

采用分子对接、网络分析预测热毒宁注射液抗流感病毒的分子作用机制, 并通过已建立的体外流感病毒神经氨酸酶筛选模型对网络预测结果验证. 结果表明, 热毒宁注射液所含化合物在化学空间上具有类药性质; 网络分析揭示出热毒宁注射液是通过与流感病毒吸附、脱壳、复制以及释放等环节的多个蛋白相互作用发挥抗流感病毒作用的; 对于预测的15 个活性分子而言, 实验结果初步证实, 对A型流感病毒, 木犀草素呈现较强的抑制作用, 槲皮素则呈现较弱的抑制作用, 这也初步证实了预测结果.     

Recognition of sialylated poly-N-acetyllactosamine chains on N- and O-linked glycans by human and avian influenza A virus hemagglutinins

Human influenza viruses are proposed to recognize sialic acids (pink diamonds) on glycans extended with poly-LacNAc chains (LacNAc=(yellow circle+blue square)). N- and O-linked glycans were extended with different poly-LacNAc chains with α2-3- and α2-6-linked sialic acids recognized by human and avian influenza viruses, respectively. The specificity of recombinant hemagglutinins (receptors in green) was investigated by using glycan microarray technology.     

Rapid Identification of the Receptor‐Binding Specificity of Influenza A Viruses by Fluorogenic Glycofoldamers

The re‐emergence of influenza raises a global concern that viral pandemics can unpredictably occur. However, effective approaches that can probe the infection risk of influenza viruses for humans are rare. In this work, we develop a glycofoldamer that can rapidly identify the glycan‐receptor specificity of influenza viruses in a high‐throughput manner. The coupling of glycan receptors that can be recognized by hemagglutinin (a surface protein on the virion capsid of influenza) to a fluorogenic‐dye foldamer produces the glycofoldamers with minimal fluorescence in aqueous solution. After interaction with human‐infecting virus strains for only five minutes, the fluorescence intensity of the glycofoldamer is remarkably enhanced with a blue‐shifted emission peak. The probes have also proven effective for the rapid identification of 1) the human‐ or bird‐infecting properties of influenza viruses in a high‐throughput manner and 2) the receptor‐specificity switch of a virus strain by mutations.     

Inhibition of influenza virus sialidase and anti-influenza virus activity by plant flavonoids

Flavonoids (103 species) were tested for inhibitory activity against influenza virus sialidase using sodium p-nitrophenyl-N-acetyl-alpha-D-neuraminate as substrate. 5,7,4'-Trihydroxy-8-methoxyflavone from the root of Scutellaria baicalensis showed the most potent activity (IC50, 55 microM), and this flavone appeared to be a non-competitive inhibitor of the enzyme. Whereas, negligible or weak inhibitory activities were observed for mouse liver sialidase, beta-galactosidase and alpha-mannosidase as tested. This flavone also inhibited the infection by influenza virus A/PR/8/34 of Madin-Darby canine kidney cells, and replication of the virus in the allantoic sack of embryonated egg. These results suggest that flavone, which has potent influenza virus sialidase inhibitory activity, may have anti-influenza virus activity.     

Antibody-scanning and epitope-tagging methods; molecular mapping of proteins using antibodies

Because synthetic short peptides bearing critical binding residues, can chemically mimic the folded antigenic determinants on proteins, short synthetic peptides can generate antibodies that react with cognate sequences in intact folded proteins. According to this mimotope theory, we produced site-specific antibodies by immunization with short peptides which overlapped each other and covered the entire protein, and used them for domain mapping of influenza virus RNA polymerase (antibody-scanning method). We also used a tagged-epitope and its monoclonal antibodies for topology mapping of clathrin light chains in clathrin triskelions by electron microscopy. Both methods using specific epitopes in combination with their antibodies enable us to determine the domains of interesting proteins systematically without the need to generate monoclonal antibodies or mutant proteins.     

The evolution and future of influenza pandemic preparedness

The influenza virus is a global threat to human health causing unpredictable yet recurring pandemics, the last four emerging over the course of a hundred years. As our knowledge of influenza virus evolution, distribution, and transmission has increased, paths to pandemic preparedness have become apparent. In the 1950s, the World Health Organization (WHO) established a global influenza surveillance network that is now composed of institutions in 122 member states. This and other surveillance networks monitor circulating influenza strains in humans and animal reservoirs and are primed to detect influenza strains with pandemic potential. Both the United States Centers for Disease Control and Prevention and the WHO have also developed pandemic risk assessment tools that evaluate specific aspects of emerging influenza strains to develop a systematic process of determining research and funding priorities according to the risk of emergence and potential impact. Here, we review the history of influenza pandemic preparedness and the current state of preparedness, and we propose additional measures for improvement. We also comment on the intersection between the influenza pandemic preparedness network and the current SARS-CoV-2 crisis. We must continually evaluate and revise our risk assessment and pandemic preparedness plans and incorporate new information gathered from research and global crises.Subject terms: Influenza virus, Infectious diseases     

Mineralized State of the Avian Influenza Virus in the Environment

Although the circulation of avian influenza viruses in humans is limited, they can be transmitted from Aves (birds) to humans, representing a great challenge. Herein, we suggest that influenza viruses from Aves might exist in a mineralized state owing to the high calcium concentrations in the avian intestine. Using two typical influenza viruses as examples, we demonstrate that these viruses can self‐mineralize in simulated avian intestinal fluid, resulting in egg‐like virus–mineral structured composites. The mineralized viruses are more robust, with enhanced infectivity and thermostability. More importantly, the mineral exterior of mineralized viruses can alter their cell internalization, expanding the possible tropisms. The discovery of a mineralized state of influenza viruses highlights the integration of nanomaterials and viruses in the environment, which provides a new understanding of avian influenza infection and its control.